Retroviral vector systems, such as lentiviral vector systems, have been proposed as a delivery system for inter alia the transfer of a nucleotide of interest to one or more sites of interest. Indeed, the concept of using viral vectors for gene therapy is well known (Verma and Somia (1997) Nature 389:239-242). Retrovirus genomes contain accessory genes, such as a rev gene, a tat gene, a vif gene, a nef gene, a vpr gene or an S2 gene. The deletion of such accessory genes, particularly when using retroviral vector systems in gene therapy is highly advantageous. Firstly it permits vectors to be produced without genes normally associated with disease in retroviral (e.g. HIV) infections. Secondly, the deletion of accessory genes permits the vector to package more heterologous DNA. Thirdly, genes whose function is unknown such as dUTPase and S2, may be omitted, thus reducing the risk of causing undesirable effects. We have previously taught, e.g. in our WO98/17815, how to remove many of the accessory genes. Further in our WO99/45126 we describe codon optimisation of the gag-pol sequence as a means of seeking to overcome the Rev/RRE requirement for export and to enhance RNA stability. However, the need remains to provide strategies for the provision of useful and safe viral vectors, and efficient means for their production.
For many gene transfer applications involving viral vectors, transduction of more than one gene is needed, and inconsistent results are sometimes obtained when coexpressing two transgenes linked by an internal ribosomal entry site (IRES) in a single bicistronic lentiviral vector (LV) transcript. Yu and coworkers described bicistronic LVs containing a gene of interest followed by an IRES and the GFP reporter gene that failed to emit detectable GFP fluorescence, possibly due to promoter interference (Yu, et al. (2003) Mol. Ther. 7: 827-38). Using a single LV containing two constitutive promoters, strong and sustained expression of both transgenes in transduced engrafting CD34(+) HSCs and their progeny was achieved, as well as in other human cell types. Thus, such dual-promoter LVs were found to coexpress multiple transgenes efficiently in a single target cell. However, it was heretofore unknown whether the presence of three promoters would be able to consistently and efficiently co-express multiple transgenes.
The present invention addresses these problems and particularly advantageously aims to provide a safer system in which viral accessory genes, such as rev, are not required in either the viral vector particle, which is used in treatment, or in their production. Further, the present invention addresses a need in the art for viral vectors that advantageously comprise three promoters that do not exhibit promoter interference and efficiently co-express multiple transgenes.